The aim of this research was to establish the optimal conditions for culture of bovine embryos obtained in vitro and the effects of osmolarity on development and apoptosis. The embryos obtained by in vitro fertilization (IVF) procedure were divided into two different groups. The embryos from the control group were cultured for 7 days in B2-Menezo. The embryos from the experimental group were cultured in medium with NaCl, having variation of concentration and osmolarity. Results showed that there are differences in development stage and apoptosis. Increasing the concentration from 90 to 120mM NaCl generated decreasing of the blastocyst stage (P&lt;0.05), and also the apoptosis was increased in the blastocyst stage. The optimum concentration of NaCl was 90mM and the optimum osmolarity of the medium was between 260-280 mOsmol for the studied IVF bovine embryos, affecting positively the embryos development

Ahmadi, Mirela

Hutu, Ioan

Milovanov, Cornelia

Mircu, Calin

Patras, Irina

Radulov, Isidora

Tulcan, Camelia

English

The aim of this research was to establish the optimal conditions for culture of bovineÂ embryos obtained in vitro and the effects of osmolarity on development and apoptosis. TheÂ embryos obtained by in vitro fertilization (IVF) procedure were divided into two differentÂ groups. The embryos from the control group were cultured for 7 days in B2-Menezo. TheÂ embryos from the experimental group were cultured in medium with NaCl, having variationÂ of concentration and osmolarity. Results showed that there are differences in developmentÂ stage and apoptosis. Increasing the concentration from 90 to 120mM NaCl generatedÂ decreasing of the blastocyst stage (P&lt;0.05), and also the apoptosis was increased in theÂ blastocyst stage. The optimum concentration of NaCl was 90mM and the optimumÂ osmolarity of the medium was between 260-280 mOsmol for the studied IVF bovineÂ embryos, affecting positively the embryos development

Annals of the University of Craiova - Agriculture, Montanology, Cadastre Series

Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru (Annals of the University of Craiova - Agriculture,Montanology, Cadastre Series) Vol. XLV 2015203OSMOLARITY AND APOPTOSIS IN IVF BOVINE EMBRYOSCORNELIA MILOVANOV1, CĂLIN MIRCU1, MIRELA AHMADI1, CAMELIA TULCAN1,ISIDORA RADULOV 2, IRINA PATRAŞ1, IOAN HUŢU1Correspondent author: cmilovanov@yahoo.comHoria Cernescu Research Unit Faculty of Veterinary Medicine1, University of Agricultural Sciencesand Veterinary Medicine of Banat “The King of Michael I” of Romania (USAMVB), Faculty ofAgriculture2 – USAMVB, Timisoara – 300645, Calea Aradului, nr. 119, RomaniaKey words: osmolarity, apoptosis, bovine, IVFABSTRACTThe aim of this research was to establish the optimal conditions for culture of bovineembryos obtained in vitro and the effects of osmolarity on development and apoptosis. Theembryos obtained by in vitro fertilization (IVF) procedure were divided into two differentgroups. The embryos from the control group were cultured for 7 days in B2-Menezo. Theembryos from the experimental group were cultured in medium with NaCl, having variationof concentration and osmolarity. Results showed that there are differences in developmentstage and apoptosis. Increasing the concentration from 90 to 120mM NaCl generateddecreasing of the blastocyst stage (P<0.05), and also the apoptosis was increased in theblastocyst stage. The optimum concentration of NaCl was 90mM and the optimumosmolarity of the medium was between 260-280 mOsmol for the studied IVF bovineembryos, affecting positively the embryos development.INTRODUCTIONSince 1967 Potts and Wilson studied the causes of cells dead in mammalianembryos, and in 1972 Kerr and his collaborators described that the cell die byprogrammed process, called apoptosis – process that assure the regulation of the cellpopulation in the development stages.Generally, the cell dead can happened by apoptosis and necrosis – which aremorphologically different. While the apoptosis affects only single cells, the necrosis acts ona large cells group. Apoptosis is not associated with any inflammatory processes, andentail consecutive distinct stages of morphology. At the beginning, it can be observed theaggregate of chromatin forming a large granular compact mass on the inner nuclearmembrane. Then, the cytoplasm is condensed and its membrane and the nuclearmembrane become extremely indented.Also, the embryo culture medium is influenced by osmolarity. As well, the culturemedium depends on salt concentration, amino acids content that modifies the osmolarityand cell volume regulation (Liu and Foote, 1996).These experiments were performed on different animal species (porcine, rabbit,mice, bovine). Liu and Foote (1995) reported their results and they showed that theconcentration of sodium chloride solution can not be higher then 93mM for rabbits. As well,the osmolarity of the cultured medium has to be 250-270mOsmol for bovine IVF embryos(Liu and Foote, 1996).Another experiment presents the cultivation of porcine IVF and NT embryos inmedium with various osmolarity. Their results presented that high osmolarity or mediumearlier cultured stage had low apoptosis rate compared to the embryos cultured in normalosmolarity (Hwang, 2008).Preimplantation embryos will develop normally in medium with low saltconcentration and low osmolarity. A higher osmolarity of embryo culture medium can allowAnalele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru (Annals of the University of Craiova - Agriculture,Montanology, Cadastre Series) Vol. XLV 2015204embryo development when there are present some organic osmolytes (for example: aminoacids) – Baltz, 2012.MATERIALS AND METHODSIn Vitro Production of EmbryoCumulus-oocyte complexes (COC) were obtaining by aspirating follicle from 1 to 5mm. The ovaries bovine were collected of slaughtered cows. The COC only with compactnon-atretic cumulus oophorus and graduated cytoplasm were selected for maturation for24 h at 39 grade C and 5% CO2 in TCM-199 supplemented with 10% fetal bovine serum(FBS).The oocytes were inseminated with semen sample from a single bull of provenfertility and layered on a discontinuous Percoll gradient (45:90) and centrifuged at 1800 gfor 30 min (Suthar and Shah, 2009).Gametes were coincubated for 18 h under the same conditions as described foroocyte maturation. Presumptive zygotes were cultured in B2-Menezo under mineral oil for7 day for all experiments on blastocysts at 39 grade C and 5% CO2.Embryos obtained by in vitro fertilization (IVF) procedures were divided in twogroups. Control groups embryos were cultured in B2-Menezo. Other group was cultured inmedium with NaCl in concentration of 90 to 120 mM and osmolarity of the medium is 260-280-mOsmol.Embryo stages after fertilization were selected (Van Soom et al.1997):two-cell, 31h;4-cell, 38h; 5 to 8 cell, 49 h; 9 to 16-cell, 100h; morula,128h; early blastocyst, day 6;expanded day 7; hatched blastocyst, day 8.A Tunel assay was used to assess the presence of apoptotic cells. Day 7 embryoswere fixed and permeabilized as described for mouse embryos (Brison and Schultz, 1996;1997) but with an increase in the time of permeabilization in 0.5% Triton X-100 to 180 min.Total number of cells and of apoptotic dead cells were counted.RESULTS AND DISCUSSIONSThere were some differences founded in the development and apoptosis incidenceduring the treatment with NaCl solution. Increasing of NaCl concentration from 90mM to120mM decreased blastocyst formation (P<0.05). The apoptosis was observed at 8-16cells stage of development (stage of genome activating at bovine) decreasing at themorula stage and increasing to blastocyst stage.Next table presents our results regarding the effect of sodium chloride concentrationon different stages of bovine embryos development, and also the apoptotic cell index.Totally we form two different treatments of bovine embryos and one control. After bovineIVF we add to the embryos sodium chloride solution in two different concentrations (90mMto 120mM) for the first three days, and after that cultured in B2-Menezo for the next days.For the control group we cultured the embryos in B2-Menezo for all the cultured period,without any sodium chloride supplementation. Our results of experiment showed that thesupplementation with sodium chloride is important for the development of embryos, but thepositive effect is depending on the concentration and osmolarity.Also, we evaluated the apoptotic cell index by TUNEL, and we observed that theapoptosis was smaller in NaCl supplemented embryos compared to the control. Ourresearch presented that there is a decrease of apoptotic cell index depending ofconcentration of NaCl, meaning that as great is the supplementation with sodium chlorideas small is the apoptosis cell death (2.91 for supplementation with NaCl 120mM and 3.47for NaCl 90mM). But this observation has to be correlated with the blastocyst percent.Blastocyst percent is greater in the supplementation with NaCl 90mM solution (34%)compared to the control (29%) and the other treatment (NaCl 120mM – 20% blastocyst).Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru (Annals of the University of Craiova - Agriculture,Montanology, Cadastre Series) Vol. XLV 2015205This happened even the apoptotic cell index is also greater in supplementation with NaCl90mM compared to the other treatment (NaCl 120mM).As well, apoptosis process is depending to the embryos quality (Van Soom et al.,1997). In the presence of fetal bovine serum (FBS) the apoptosis cell death is increased(Byrne et al., 1999). However, the rate of apoptosis is not influenced by heat inactivation offetal bovine serum – which contains a lot of nutrients for the embryos survival factors, suchas: growth factors – peptide nature, and also cytokines or tumor necrosis factor – that caninduce apoptosis process (Brison and Schultz, 1996, 1997, 1998; Suthar and Shah, 2009).Table Effects of NaCl on the development of bovine IVF embryosTreatment No. of oocytescultured% > 2 cell % blastocyst % TunelControl 76 82 29 3.88NaCl (90mM) 78 88 34 3.47NaCl (120mM) 75 74 20 2.91For a very well development of embryos, after this research, we obtain the optimumconcentration of NaCl 90 mM, and the optimal osmolarity of the medium was 260-280mOsmol. Apoptotic dead cell index in day 7 of embryo blastocyst was reversalcorrelated with the total number of cells.CONCLUSIONSConcentration optimal of NaCl is of 90 mM and osmolarity of the medium is 260-280mOsmol for bovine IVF embryos.Apoptotic dead cell index in day 7 blastocyst is correlated negatively with thenumber total of cell. The Tunel percent is greatest for bovine embryos withoutsupplementation of saline solution, and was the lowest for the embryos supplemented withsodium chloride 120mM solution.REFERENCESBaltz J.M. (2012) Media composition: salts and osmolarity, Methods Mol Bio, 921,61-80.Byrne AT, Southgate J., Brison DR, Leese HJ (1999) Analysis of apoptosis in thepreimplantation bovine embryo using TUNEL, Journal of Reproduction and fertility, 117:97-105.Brison D.R. and Schultz R.M. (1996). RT-PCR-based method to localize thespatial expression of genes in the mouse blastocyst, Molecular Reproduction andDevelopment 44:171-178Brison D.R. and Schultz R.M. (1997) Apoptosis during mouse blastocyst formationfor: evidence for a role for survival factors including TGFF-ALFA. Biology of Reproduction56:1088-1096Brison D.R. and Schultz R.M. (1998) Increased incidence of apoptosis intransforming growth factor -deficient mouse blastocyst, Biology of Reproduction, 59: 136-144.Hwang IS, Park MR, Moon HJ, Shim JH, Kim DH, Yang BC, Ko YG, Yang BS,Cheong HT, Im GS (2008) Osmolarity at early culture stage affects development andexpression of apoptosis related genes (Bax- and Bcl-xl) in pre-implantation porcine NTEmbryos, Molecular Reproduction and Development, 75: 464-471.Kerr J.F., Wyllie A.H., Currie A.R. (1972) Apoptosis: a basic biologicalphenomenon with wide ranging implications in tissue kinetics, British Journal of Cancer,26, 239-257.Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru (Annals of the University of Craiova - Agriculture,Montanology, Cadastre Series) Vol. XLV 2015206Liu Zishu, Foote Robert H. (1995) Effects of inositol and glycine with increasingsodium chloride and constant osmolarity on development of rabbit embryos, J. Assist.Reprod. Genet., 12: 141-146.Liu Zishu, Foote Robert H. (1996) Sodium chloride, osmolyte, and osmolarityeffects on blastocyst formation in bovine embryos produced by in vitro fertilization (IVF)and cultured in simple serum-free media, Journal of Assisted Reproduction and Genetics,13(7): 562-8.Potts D.M., Wilson I.B. (1967) The preimplantation conceptus of the mouse at 90hours post-coitum, Journal of Anatomy, 1021-11Suthar VS, Shah RG (2009) Bovine in vitro embryo production: An overview,Veterinary world, 2(12): 478-479Van Soom A, Ysebaert M and De Kruif A.(1997) Relationship between timing ofdevelopment, morula morphology and cell allocation to the inner cell mass andtrophectoderm in in vitro produced embryos. Molecular Reproduction and Development47:47-56

OSMOLARITY AND APOPTOSIS IN IVF BOVINE EMBRYOS

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OSMOLARITY AND APOPTOSIS IN IVF BOVINE EMBRYOS

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Annals of the University of Craiova - Agriculture, Montanology, Cadastre Series